PA1-16889

antibody from Invitrogen Antibodies

Targeting: ACTB

Provider product page for PA1-16889

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [29]
Comments [0]
Validations
Immunocytochemistry [1]
Immunohistochemistry [2]
Other assay [31]

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Product number: PA1-16889 - Provider product page
Provider: Invitrogen Antibodies
Product name: beta Actin Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Other
Description: Suggested positive control: Hela whole cell extract. Based on 100% sequence identity, this antibody is predicted to react with Rat, X. tropicalis, Chicken, Sheep, Bovine, Dog, Horse, Rabbit, Guinea pig, Pig, Golden hamster, Orangutan, and Chimpanzee. For IHC, epitope retrieval with citrate buffer pH6.0 is recommended for FFPE tissue sections.
Reactivity: Human, Mouse, Canine, Chicken/Avian
Host: Rabbit
Isotype: IgG
Vial size: 100 μg
Concentration: 1 mg/mL
Storage: 4°C, do not freeze

ACTB protein structure - PA1-16889 shown in red.

Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Experimental details: NULL

Supportive validation

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Supportive validation

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Supportive validation

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Experimental details: Figure 4 Relationship between the expression of miR-34a-5p and that of the miR-34a-5p targets during the HPCs commitment. ( A ) Detection of lymphoid enhancer-binding factor 1 ( LEF1 ), nuclear receptor subfamily 4, group A, member 2 ( NR4A2 ) and MYB expression levels by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) in Negative Control (NegCTR) mimic and miR-34a-5p mimic-transfected CD34+ cells at 24 h post-nucleofection; ( B ) Western Blotting analysis of Lef1 ( i ), Nr4a2 ( ii ) and Myb ( iii ) protein levels in protein lysates from miR-34a-5p mimic-transfected compared to NegCTR mimic-transfected CD34+ cells at 24 h after the last of two nucleofection cycles. Actin protein levels are reported as loading control; ( C - F ) Expression kinetics of miR34a ( i ), LEF1 ( ii ) and NR4A2 ( iii ) during the megakaryocyte ( C ); monocyte/macrophage ( D ); erythroid ( E ) and granulocyte ( F ) differentiation of CD34+ cells. miR-34a-5p, LEF1 and NR4A2 expression levels were monitored by RT-qPCR at day 4 and day 8 post-CD34+ cells purification. Modulations of miR-34a-5p, LEF1 and NR4A2 transcripts are reported as Relative Quantity (RQ) respect to freshly purified CD34+ cells (DAY 0) sample, which was set as calibrator. Data are from n = 3 independent experiments performed with different healthy donor-derived cord blood units. Values in the graph are reported as mean +- SEM. * p

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Experimental details: Figure 5 Effects of LEF1 and NR4A2 silencing on CD34+ hematopoietic progenitor cells proliferation and clonogenic activity. ( A ) Detection of LEF1 and NR4A2 expression levels by RT-qPCR in NegCTR siRNA, LEF1 siRNA and NR4A2 siRNA-transfected CD34+ cells at 24 h post-nucleofection; ( B ) Western Blotting analysis of Lef1 ( i ) and Nr4a2 ( ii ) protein levels in LEF1 siRNA ( i ) or NR4A2 siRNA ( ii ) compared to NegCTR siRNA-transfected CD34+ cells at 24 h after the last of two nucleofection cycles. Actin protein levels are reported as loading control; ( C ) Statistical analysis results (mean +- SEM; n = 3) for the percentage of cells in the different cell cycle phases performed by propidium iodide staining 24 h post-nucleofection; ( D , E ) Methylcellulose clonogenic assay results (mean +- SEM; n = 3). The colony scoring results are reported as total number of colonies grown from 200 cells plated ( D ) and percentages of each colony type ( E ); Colonies were scored according to the manufacturer's guidelines; ( F , G ) Megakaryocyte clonogenic assay results (mean +- SEM; n = 3) in terms of megakaryocyte colony number ( F ) and size ( G ). Values are reported as number of megakaryocyte colonies for 2000 plated cells. Error bars in the graphs represent SEM. * p

Supportive validation

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Experimental details: Figure 6 miR-494-3p overexpression reduces SOCS6 expression and enhances STAT3 phosphorilation A-B. Western blot analysis of SOCS6 protein levels in whole cell lysates from K562 (A) and CB CD34+ (B) cells overexpressing miR-494-3p at different time-points after mimic nucleofection. SOCS6 protein levels in miR-494-3p overexpressing cells were compared with control samples nucleofected with mimic Negative Control (Neg CTR). beta-actin was included as loading control while PTEN was selected as positive control. C. Immunoblots representing phospho-STAT3 increase in CB CD34+ cells overexpressing miR-494-3p compared with negative control (Neg CTR) 48 and 96 hours after nucleofection. beta-actin was included as loading control.

Supportive validation

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Experimental details: Figure 8 miR-494-3p inhibition impairs MK differentiation of PMF CD34+ cells A. Scatter plot representing the correlation between miR-494-3p and SOCS6 expression levels according to the microarray analysis in our initial dataset of 42 PMF samples and 31 healthy donors. The Spearman rank correlation coefficient (r) was used to measure and identify the degree of linear dependence between the ranked variables (r = -0.57, p = 1.12 x 10 -7 ). Green circles = PMF CD34+ cells, Red triangles = PB CD34+ cells, Blue squares = BM CD34+ cells. Abbreviations: PMF, primary myelofibrosis; PB, peripheral blood; BM, bone marrow; CTR, control. B. Western blot analysis of SOCS6 protein levels in PMF CD34+ cells compared to CB CD34+ cells. beta-actin was included as loading control. C-D. Expression levels of miR-494-3p, SOCS6, and PTEN in PMF CD34+ cells after miRNA inhibition evaluated by means of qRT-PCR. Data are reported as RQ mean +- S.E.M (n=3). E. Immunoblots representing SOCS6 protein levels in whole cell lysates from PMF CD34+ cells after miR-494-3p inhibition. SOCS6 protein levels were evaluated 24, 48 and 96 hours after miR-494-3p inhibitors nucleofection and were compared with control samples electroporated with Negative Control inhibitors (NegCTR). beta-actin was included as loading control while PTEN was selected as positive control. F-G. Results of flow cytometry analysis of the percentages of CD41+ (F) and CD42b+ (G) cells evaluated at days 3, 5, 8, 10 and 12 in serum-free MK uni

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Experimental details: Fig. 6 TiO 2 -NPs trigger autophagic response in HaCaT cells by forming autophagosomes and increasing LC3 translocation. Representative images of GFP-LC3 puncta. a Evaluation of LC3 puncta on transiently transfected eGFP-LC3-HaCaT cells treated with TiO 2 -NPs by confocal microscopy. The green color represents the GFP-LC3 puncta and the red color represents the cytoskeleton. Control cells were transfected with eGFP-LC3 plasmid and grown only in DMEM complete cell media and later cells were treated with 0.16 ug/mL (low-dose) and 25.0 ug/mL (high-dose) TiO 2 -NPs for 1 and 24 h before fixation. b Quantification of GFP-LC3 dots per cell ( green dots ). Up to twenty cells were analyzed. c Representative western blots probed with anti-LC3B antibody (Novus) from cells treated for 1 and 24 h; cells without treatment were used as a control. Data obtained was analyzed for the conversation of LC3I (cytoplasmatic) to LC3II (membrane-bound) by western blotting using an LC3B antibody. Actin was used as a loading control. Values represent mean +- SD. * P < 0.05, ** P < 0.01 and **** P < 0.0001. Scale bar ( bottom-left ) represents 20 um

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Experimental details: Fig 1 Design and Function of OVA and MC.OVA Vaccine Vectors. (A) Linear diagrams showing the functional features of the OVA (upper) and MC.OVA (lower) vaccine constructs. (B, C) 293 cells were transfected with 1 mug of the indicated plasmid. Lysates were probed for OVA expression by Western blot and expression was measured relative to beta-actin. (D) 293 cells transfected with NF-kappaB SEAP reporter and either GFP, OVA, or MC.OVA expression plasmids. Cells were plated with increasing concentrations of rim.

Supportive validation

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Experimental details: Fig. 5 Effect of NBP treatment on the expression of the vascular endothelial growth factor (VEGF) and angiopoietins-2 (Ang-2) proteins in tissue lysates of the injection site in a rat model of ICH. Panel ( a ) shows representative immunoblot images (upper two sets) of VEGF and Ang-2 in the groups as indicated, with representative images (bottom set) showing equal protein loading as blotted with the beta-actin antibody. Panels ( b , c ) plot the protein levels expressed as relative optic densities (o.d.) normalized to the internal control beta-actin. Results from two-way ANOVA followed by Bonferroni's post hoc tests are marked with symbols, with * indicating significantly different comparing to the Sham groups; # indicating significantly different comparing to the ICH groups; and & indicating significantly different comparing to the ICH + NBP10 groups

Supportive validation

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Experimental details: Figure 2 Basal autophagy is not altered across different stages of disease progression in R6/2. ( A )-( D ) Representative immunoblot for the expression of ATG proteins p62/SQSTM1, LC3B, GABARAPL2 (GL2) from 2, 4, 8, and 12 weeks in cortex, hippocampus, striatum and cerebellum, respectively. beta-ACTIN is used as a loading control. ( E )-( H ) Pooled quantified bar graphs for p62 for cortex: Age x Genotype (F (3,54) = 0.1322, p = 0.94), hippocampus: Age x Genotype (F (3,54) = 0.4656, p = 0.70), striatum: Age x Genotype (F (3,54) = 1.394, p = 0.25), and cerebellum: Age x Genotype (F (3,54) = 0.6080, p = 0.61) respectively. ( I )-( L ) Pooled quantified bar graphs for LC3B-I for cortex: Age x Genotype (F (3,54) = 2.380, p = 0.07); (12 weeks: *p < 0.05), hippocampus: Age x Genotype (F (3,54) = 0.1890, p = 0.90), striatum: Age x Genotype (F (3,54) = 0.09385, p = 0.96), and cerebellum: Age x Genotype (F (3,54) = 0.1832, p = 0.90) respectively. ( M )-( P ) Quantified bar graphs for LC3B-II for cortex: Age x Genotype (F (3,54) = 0.03193, p = 0.99), hippocampus: Age x Genotype (F (3,54) = 0.2341, p = 0.87), striatum: Age x Genotype (F (3,54) = 0.3311, p = 0.80), and cerebellum: Age x Genotype (F (3,54) = 0.2769, p = 0.84) respectively. ( Q )-( T ) Summary of quantified bar graphs for GABARAPL2-II (GL2) for cortex: Age x Genotype (F (3,54) = 0.5814, p = 0.62), hippocampus: Age x Genotype (F (3,54) = 0.3486, p = 0.79), striatum: Age x Genotype (F (3,54) = 0.07321, p = 0.97), and cerebe

Supportive validation

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Experimental details: Figure 5 Nilotinib (Tasigna(tm)) does not affect inducing autophagy in wild-type control mice. ( A )-( D ) Representative immunoblot for the expression of ATG proteins p62/SQSTM1, LC3B, GABARAPL2 (GL2) from 6, 8, 10 and 12 weeks in cortex, hippocampus, striatum and cerebellum, respectively. beta-ACTIN is used as a loading control. ( E )-( H ) Pooled quantified bar graphs for p62/SQSTM1 for cortex: Age x treatment (F (3,12) = 2.32, p = 0.12), hippocampus: Age x treatment (F (3,12) = 1.87, p = 0.18), striatum: Age x treatment (F (3,12) = 0.99, p = 0.42), and cerebellum: Age x treatment (F (3,12) = 5.40, p = 0.01); (8 weeks: *p < 0.01) respectively. ( I )-( L ) Pooled quantified bar graphs for LC3B-I for cortex: Age x treatment (F (3,12) = 0.59, p = 0.63), hippocampus: Age x treatment (F (3,12) = 1.72, p = 0.21), striatum: Age x treatment (F (3,12) = 1.13, p = 0.37), and cerebellum: Age x treatment (F (3,12) = 3.06, p = 0.06) respectively. ( M )-( P ) Pooled quantified bar graphs for LC3B-II for cortex: Age x treatment (F (3,12) = 1.65, p = 0.22), hippocampus: Age x treatment (F (3,12) = 0.57, p = 0.64), striatum: Age x treatment (F (3,12) = 1.22, p = 0.34), and cerebellum: Age x treatment (F (3,12) = 2.22, p = 0.13) respectively. ( Q )-( T ) Pooled quantified bar graphs for GABARAPL2-II (GL2) for cortex: Age x treatment (F (3,12) = 1.86, p = 0.18); (10 weeks: *p < 0.05), hippocampus: Age x treatment (F (3,12) = 1.01, p = 0.42), striatum: Age x treatment (F (3,12) = 0.08, p = 0.

Supportive validation

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Experimental details: Figure 6 Nilotinib (Tasigna(tm)) is ineffective in inducing autophagy across different stages of disease progression in R6/2. ( A )-( D ) Representative immunoblot for the expression of ATG proteins p62/SQSTM1, LC3B, GABARAPL2 (GL2) in cortex, hippocampus, striatum and cerebellum respectively from 6, 8, 10 and 12 weeks. beta-ACTIN is used as a loading control. ( E )-( H ) Pooled quantified bar graphs for p62/SQSTM1 for cortex: Age x treatment (F (3,12) = 2.29, p = 0.12), hippocampus: Age x treatment (F (3,12) = 0.28, p = 0.83), striatum: Age x treatment (F (3,12) = 0.39, p = 0.76), and cerebellum: Age x treatment (F (3,12) = 0.27, p = 0.84) respectively. ( I )-( L ) Pooled quantified bar graphs for LC3B-I for cortex: Age x treatment (F (3,12) = 3.27, p = 0.05), hippocampus: Age x treatment (F (3,12) = 0.27, p = 0.84), striatum: Age x treatment (F (3,12) = 0.85, p = 0.49), and cerebellum: Age x treatment (F (3,12) = 0.55, p = 0.65) respectively. ( M )-( P ) Summary of quantified bar graphs for LC3B-II for cortex: Age x treatment (F (3,12) = 0.91, p = 0.46), hippocampus: Age x treatment (F (3,12) = 0.13, p = 0.93), striatum: Age x treatment (F (3,12) = 4.68, p = 0.02), and cerebellum: Age x treatment (F (3,12) = 0.43, p = 0.73) respectively. ( Q )-( T ) Pooled quantified bar graphs for GABARAPL2-II (GL2) for cortex: Age x treatment (F (3,12) = 3.30, p = 0.05), hippocampus: Age x treatment (F (3,12) = 0.20, p = 0.88), striatum: Age x treatment (F (3,12) = 3.09, p = 0.06), and ce

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Experimental details: Figure 6 Western blot analysis of Eml1 mutant retinas. Graphs showing optical density of Western blot bands against amount of total retina protein, n = 3. Eml1 mutant retinas (empty circles) as compared to the wild type retinas (filled circles). Linearity of plots demonstrates sub-saturating ECL signal ensuring direct quantitative comparison. ( A ) Actin, ( B ) EML1, ( C ) Rhodopsin, Rh, ( D ) transducin alpha, Gtalpha, ( E ) transducin gamma, Gtgamma, and ( F ) phosphodiesterase gamma (PDEgamma). Representative staining for each protein is shown in insets (for complete blot pictures see supplementary Fig. 4 ).

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Experimental details: Fig. 2 EBI2 deficiency results in transient delay in MBP expression. a Representative confocal images show mice cerebellar slices prepared from P10 mice and cultured for 1, 2, 4 to 5, 7 to 9, and 14 DIV. Myelin (MBP, green) staining is shown. Myelin development was determined by quantification of MBP staining density. Analysis showed significant differences in myelin development between one to two DIV. Images taken at x10 magnification. Scale bar, 100 muM. b Graph showing quantification of MBP immunofluorescence in images such as those shown in ( a ). Data presented as mean +- SD (five to six animals per group), unpaired t test, ** p < 0.01, *** p < 0.001 versus corresponding control. c Representative WBs show differential expression of MBP in whole brain lysates prepared from embryonic (E14 and E18) and postnatal (P1, P7, P14, and P30) WT and EBI2 KO mice. d, e Densitometric quantification of blots such as that shown in ( c ). Normalized WB data presented as mean +- SD (three to four WB data per time point; one animal per blot), unpaired t test, *** p < 0.001 versus corresponding control

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Experimental details: Fig. 1 Erastin treatment promotes ubiquitin-proteasomal degradation of VDAC2/3. a A375 cells expressing GFP-VDAC2 or GFP-VDAC3 and mCherry-VDAC1 were treated with erastin (5 uM). Representative images were taken at the indicated time after treatment. Scale bars, 10 mum. Fluorescence intensity was assayed using Image J software. b A375 cells were treated with erastin at the indicated time (0-12 h, 10 uM) or indicated concentrations (0-10 uM, 12 h). Western blot analysis shows the expression of VDAC2, VDAC3, and VDAC1 in indicated cells. c , d Knockdown of VDAC2 and VDAC3 individually or in combination suppressed erastin-induced ferroptotic cell death. Indicated cells were treated with erastin (5 uM) for 24 h. Cell viability was assayed using a CCK8 kit. The lipid formation was measured by MDA assay. The accumulation of Fe 2+ was measured by iron detection assay. The concentrations of GSH and GSSG were detected by relative assay kits ( c ). The protein level of VDAC2/3 was assessed by immunoblotting ( d ). e , f Overexpression of VDAC2 and VDAC3 together increased erastin-induced ferroptotic cell death. Indicated cells were treated with erastin (5 uM) for 24 h. Cell viability, intracellular MDA, Fe 2+ , GSH, and GSSG levels were measured ( e ). The overexpression of VDAC2/3 was assessed by immunoblotting ( f ). g A375 cells were treated with erastin (5 uM) for 8 h, then MG132 (50 mM) or NH 4 Cl (50 mM) were added into the culture medium for 4 h. The protein level of VDAC2/3 was

Supportive validation

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Experimental details: Fig. 4 Nedd4 regulates erastin-induced ferroptosis. a - f Knockdown of Nedd4 enhanced erastin-induced ferroptotic cell death. Indicated cells were treated with erastin (1-20 uM) for 24 h, and cell viability was assayed using a CCK8 kit ( a ). Indicated cells were treated with erastin (5 uM) for 24 h, the lipid ROS level was assessed by flow cytometry using C11-BODIPY ( b ), and the lipid formation was measured by MDA assay ( c ). The accumulation of Fe 2+ was measured by iron detection assay ( d ), concentrations of GSH, and GSSG were detected by relative assay kits ( e ). The expression of Nedd4 was assessed by immunoblotting and compared to actin levels ( f ). Number 1 and black color represent control sh group; number 2 and red color represent Nedd4 sh + Vector group; number 3 and green color represent Nedd4 sh + Nedd4 WT group; number 4 and blue color represent Nedd4 sh + Nedd4 C867S group. g - l Overexpression of Nedd4 suppressed erastin-induced ferroptotic cell death. Indicated cells were treated with erastin (1-20 uM) for 24 h, and cell viability was assayed using a CCK8 kit ( g ). Indicated cells were treated with erastin (5 uM) for 24 h, the lipid ROS level was assessed by flow cytometry using C11-BODIPY ( h ), and the lipid formation was measured by MDA assay ( i ). The accumulation of Fe 2+ was measured by iron detection assay ( j ), concentrations of GSH and GSSG were detected by relative assay kits ( k ). The expression of Nedd4 was assessed by immunoblotting and

Supportive validation

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Experimental details: Fig. 5 Nedd4 regulates VDAC2/3 degradation through specific ubiquitination sites. a A375 cells were transfected with combinations of DNA constructs as indicated and treated with MG132 for 4 h. Lysates were immunoprecipitated with anti-Myc, and western blots were performed to analyze the presence of indicated proteins and levels of ubiquitination. b A375 cells were transfected with combinations of DNA constructs as indicated and treated with erastin (5 uM) for indicated time. Cell lysates were analyzed by immunoblotting with indicated antibodies. The protein level of VDAC2/3 was quantitated by Image J and normalized to actin levels. c - e Overexpression of KR mutants of VDAC2/3 partially rescued the cell sensitivity to erastin, which was inhibited by Nedd4. A375 cells were transfected with indicated DNA constructs for 48 h and treated with erastin (5 uM) for 12 h. Cell viability, intracellular MDA, and GSH levels were measured ( c ). The lipid ROS level was assessed by flow cytometry using C11-BODIPY ( d ). The protein levels of overexpressed constructs were assessed by immunoblotting ( e ). f Quantification of MMP during erastin-induced ferroptosis. A375 cells were treated with erastin and 50 nM TMRE was added 20 min before each indicated time point. g Suppression of VDAC2/3 inhibited ferroptosis-associated MMP hyperpolarization. A375 cells expressing indicated shRNA constructs were treated with erastin and 50 nM TMRE was added 20 min before each indicated time point. h Overe

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Experimental details: Fig. 3 CBX2 depletion induces proliferation arrest and apoptosis in advanced PCa cell lines. a , b Confirmation of CBX2 mRNA knockdown in LNCaP and C4-2 cells by qPCR; c , d Confirmation of CBX2 protein knockdown in LNCaP and C4-2 cells; e , f MTT analysis of cell viability following CBX2 silencing in LNCaP and C4-2 cells; g , h Assessment of caspase 3-7 activity in LNCaP and C4-2 cells following CBX2 depletion

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Experimental details: Fig. 4 Antidepressants-mediated effect on cellular protein content. HT-22 cells were treated with antidepressants for 48 and 96 h and densitometry analysis of NF-kappaB ( b ), p16 ( c ), p21 ( d ), p27 ( e ), p53 ( f ), TRF1 ( g ), TRF2 ( h ), calnexin ( i ), NuMa ( j ), cleaved caspase 3 ( k ), Bcl-2 ( l ) was evaluated. Representative Western Blots are presented ( a ). Bars indicate SD, n = 3, *** /^^^ p < 0.001, ** /^^ p < 0.01, * /^ p < 0.05, no indication--no statistical significance (one-way ANOVA and Dunnett's a posteriori test)

Supportive validation

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Experimental details: Figure 1 pDNA(plasmid DNA)-mediated klotho overexpression in human monocytic cells Cells were transfected with pDNA and selected in antibiotics to obtain stable clones and then Western Blot analysis of klotho membrane (130 kDa) and secrete (65 kDa) forms expression was performed ( A ). Representative images of Western Blot membrane and Coomassie staining are presented ( B ). The bands were quantified and normalized to their corresponding beta-actin bands in the case of membrane form of klotho or to CBB staining in the case of secrete form. Bars indicate SD, n = 3, *** p < 0.001, ** p < 0.01 (one-way ANOVA and Dunnett's a posteriori test). ACTB actin, KLTH klotho, CBB Coomassie Brilliant Blue staining.

Supportive validation

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Experimental details: Figure 2 Expression of proteins involved in the Golgi stress response. Klotho expression was upregulated with pDNA, cells were treated for 72 h with monensin sodium or 168 h with LPS and expression of proteins involved in CREB34L-, HSP47- and TFE3-mediated pathways was evaluated with Western Blot method (A), i.e.,: CREB34L (B), ARF4 (C), HSP47 (D), and TFE3 (E). The bands were quantified and normalized to their corresponding β-actin bands. Bars indicate SD, n = 3, *** p < 0.001, ** p < 0.01, * p < 0.05, no indication - no statistical significance (one-way ANOVA and Dunnett’s a posteriori test).

Supportive validation

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Experimental details: Figure 3 Expression of proteins involved in the Golgi-mediated stress response. Klotho expression was upregulated with pDNA, cells were treated for 72 h with monensin sodium or 168 h with LPS and the expression of TFE3-related proteins was evaluated with Western Blot method ( A ), i.e.,: SIAT4A ( B ), GRASP65 ( C ), WIPI1 ( D ), GCP60 ( E ), and giantin ( F ). The bands were quantified and normalized to their corresponding beta-actin bands. Bars indicate SD, n = 3, *** p < 0.001, ** p < 0.01, * p < 0.05, no indication - no statistical significance (one-way ANOVA and Dunnett's a posteriori test).

Supportive validation

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Experimental details: Figure 4 Expression of factors involved in ER stress response. Klotho expression was upregulated with pDNA, cells were treated for 72 h with monensin sodium or 168 h with LPS and expression of proteins involved in ATF6- and IRE1-mediated pathways was evaluated with Western Blot method ( A ), i.e.,: c-ATF6 ( B ), MAPK ( C ), p-IRE1 ( D ), p-ASK1 ( E ), and TRAF2 ( F ). XBP-1 splicing was evaluated by RT-PCR ( G ) and O-GlcNAc levels by WB ( H , I ). The bands were quantified and normalized to their corresponding beta-actin bands. Bars indicate SD, n = 3, *** p < 0.001, ** p < 0.01, * p < 0.05, no indication - no statistical significance (one-way ANOVA and Dunnett's a posteriori test).

Supportive validation

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Experimental details: Figure 5 Expression of factors involved in ER stress response. Klotho expression was upregulated with pDNA, cells were treated for 72 h with monensin sodium or 168 h with LPS and expression of proteins involved in PERK-mediated pathway was evaluated with Western Blot method ( A ), i.e.,: p-PERK ( B ), ATF4 ( C ), p-eIF2alpha ( D ), CHOP ( E ), and GADD34 ( F ). The bands were quantified and normalized to their corresponding beta-actin bands. Bars indicate SD, n = 3, *** p

Supportive validation

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Experimental details: Fig. 4 AGC3 activates NF-kappaB (p65) and MAPK (p38) in RAW 264.7 Macrophages. RAW 264.7 macrophages were treated with AGC3 (12.5 mug/mL), LPS (1 mug/mL), or culture medium for 15 min. Total protein was extracted using RIPA buffer and changes in phosphorylated p65 (phospho-p65) and phosphorylated p38 (phospho-p38) were analyzed using Western blots and densitometry. beta-actin was used as a loading control. (A) Western blot of p65 and p38 activation; (B) Ratio of p65:beta-actin; (C) Ratio of phospho-p65:beta-actin; (D) Ratio of p38:beta-actin; (E) Ratio of phospho-p38:beta-actin. Results are shown as the mean of the ratio for each protein to beta-actin +- SEM for three independent treatments. Densitometry values were determined using ImageJ and compared (untreated versus LPS; untreated versus AGC3) by non-parametric Mann-Whitney's U test (* p